Journal: Journal of Translational Medicine

Article Title: Network medicine modeling of the m⁶A regulatory landscape identifies a KLF6–WTAP axis as a therapeutic target in pulmonary fibrosis

doi: 10.1186/s12967-026-07876-x

Figure Lengend Snippet: WTAP promotes fibroblast proliferation and migration in pulmonary fibrosis through m⁶A methylation. ( A ) Representative immunofluorescence images of the merged photos in normal and IPF samples. ( B ) Representative western blot images of WTAP. ( C ) qRT-PCR result of the transfection efficiency. ( D ) Knockdown of WTAP significantly reduced the expression of α-SMA and Collagen III in TGF-β–stimulated fibroblasts and reversed their upregulation induced by TGF-β. ( E ) Representative immunofluorescence images to detect WTAP, Ki-67 and α-SMA expression in TGF-β-induced groups at 48 h. Scale bars, 20 μm. ( F ) Cell proliferation was measured by EdU staining. ( G - H ) Fibroblasts migration ability was measured by the wound healing and transwell assay. ( I ) Dot blot assay using an anti-m⁶A antibody in TGF-β-induced fibroblasts, shWTAP and NC groups. MB staining was included as a loading control. P < 0.05 was considered significant. ( J ) Western blot images of IGFBP5, KLF6, NR4A3 and MYC levels. ( K ) MeRIP-qPCR analysis of MYC, IGFBP5, KLF6 and NR4A3 in TGF-β-induced fibroblasts. ( L ) Putative KLF6 binding site on the promoter region of WTAP. ( M ) KLF6 binds to the promoter region of WTAP in TGF-β induced fibroblasts. Chromatin-IP was performed using KLF6 antibody or control IgG. Values are percentage of input. ( O ) KLF6 activates WTAP promoter activity in dual-luciferase reporter assay. ( O ) Western blotting analysis and correlation analyses of WTAP expression with expression of KLF6 in 8 freshly collected human IPF samples. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to the corresponding groups

Article Snippet: The membranes were blocked with 5% bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies against WTAP (Proteintech, Cat No. 60188-1-Ig), Collagen III(Proteintech, Cat No. 22734-1-AP), KLF6 (Proteintech, Cat No. 14716-1-AP), NR4A3 (Proteintech, Cat No. 55405-1-AP), MYC(Proteintech, Cat No. 10828-1-AP), GAPDH (Proteintech, Cat No. 60004-1-Ig), α-SMA (Proteintech, Cat No. 14395-1-AP).

Techniques: Migration, Methylation, Immunofluorescence, Western Blot, Quantitative RT-PCR, Transfection, Knockdown, Expressing, Staining, Transwell Assay, Dot Blot, Control, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Luciferase, Reporter Assay